Even at the present time when medical science has advanced, the 5-year survival rate of cancer patients, particularly solid tumor patients (other than blood cancer patients) who account for the majority of cancer patients is less than 50%. About two-thirds of all cancer patients are diagnosed at a progressed stage, and most of them die within 2 years after cancer diagnosis. Such poor results in cancer therapy are due not only to the problem of therapeutic methods, but also to the fact that it not easy to diagnose cancer at an early stage and to accurately diagnose progressed cancer and to carry out the follow-up of cancer patients after cancer therapy.
In current clinical practice, the diagnosis of cancer is confirmed by performing tissue biopsy after history taking, physical examination and clinical assessment, followed by radiographic testing and endoscopy if cancer is suspected. However, the diagnosis of cancer by the existing clinical practices is possible only when the number of cancer cells is more than a billion and the diameter of cancer is more than 1 cm. In this case, it is reported that the cancer cells already have metastatic ability, and at least half thereof have already metastasized. Meanwhile, tumor markers for monitoring substances that are directly or indirectly produced from cancers are used in cancer monitoring after the treatment of the cancer, but they have limitations in early diagnosis, since up to about half thereof appear normal even in the presence of cancer, and they often appear positive even in the absence of cancer.
The reason why the early diagnosis and treatment of cancer are difficult is that cancer cells significantly differ from normal cells and are highly complex and variable. Cancer cells grow excessively and continuously, invading surrounding tissue and metastasize to distal organs leading to death. Despite the attack of an immune mechanism or anticancer therapy, cancer cells survive and continually develop, and cell groups that are most suitable for survival selectively propagate. Cancer cells are living bodies with a high degree of viability, which occur by the mutation of a large number of genes. In order that one cell is converted to a cancer cell and developed to a malignant cancer lump that is detectable in clinics, the mutation of a large number of genes must occur. Thus, in order to diagnose and treat cancer at the root, approaches at a gene level are necessary.
Recently, genetic analysis has been actively attempted to diagnose cancer. The simplest typical method is to detect the presence of ABL: BCR fusion genes (the genetic characteristic of leukemia) in blood by PCR. The method has an accuracy rate of more than 95%, and after the diagnosis and therapy of chronic myelocytic leukemia using this simple and easy genetic analysis, this method is being used for the assessment of the result and follow-up study. However, this method has the deficiency that it can be applied only to some blood cancers.
Furthermore, another method has been attempted, in which the presence of genes expressed by cancer cells is detected by RT-PCR and blotting, thereby diagnosing cancer cells present in blood cells. However, this method has shortcomings in that it can be applied only to some cancers, including prostate cancer and melanoma, has a high false positive rate. In addition, it is difficult to standardize detection and reading in this method, and its utility is also limited (Kopreski, M. S. et al., Clin. Cancer Res., 5:1961, 1999; Miyashiro, I. et al., Clin. Chem., 47:505, 2001).
Particularly, it is known that fragmented DNAs are released from abnormal cells in the cancer tissue of cancer patients into blood flow by processes including apoptosis and necrosis, and thus exist as cell-free tumor DNA in the serum or plasma of the blood. In fact, it is known that there are frequent cases in which the concentration of DNA in the blood flow of many cancer patients is higher than that in normal people. Cancer-specifically abnormally modified DNA regions have been attempted to be used as markers for diagnosing cancer (deVos T. et al., Clin. Chem. 55:1337-1346; Potter N. T. et al., Clin. Chem. 60:9; Messaoudi S. EI. et al., Clinica Chimica Acta 424:222-230; Marzese D. M. et al., Expert Rev. Mol. Diagn. 13(8):827-844).
Actually, there has been an active attempt to diagnose lung cancer, head and neck cancer, breast cancer, colon cancer, and liver cancer by examining the promoter methylation of mutated K-Ras oncogenes, p53 tumor-suppressor genes and p16 genes in serum, and the labeling and instability of microsatellite (Chen, X. Q. et al., Clin. Cancer Res., 5:2297, 1999; Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedes, M. et al., Cancer Res., 60:892, 2000; Sozzi, G. et al., Clin. Cancer Res., 5:2689, 1999).
Meanwhile, in samples other than blood, the DNA of cancer cells can also be detected. A method has been attempted in which the presence of cancer cells or oncogenes in sputum or bronchoalveolar lavage of lung cancer patients is detected by a gene or antibody test (Palmisano, W. A. et al., Cancer Res., 60:5954, 2000; Sueoka, E. et al., Cancer Res., 59:1404, 1999). Additionally, another method of detecting the presence of cancer cells-derived modified genes in mucosal cells in feces of colon and rectal cancer patients (Ahlquist, D. A. et al., Gastroenterol., 119:1219-27, 2000) has been attempted. However, since these gene mutations occur in some cancer patients, there is a limitation in accurately performing the early diagnosis of cancers.
In the genomic DNA of mammalian cells, there is the fifth base in addition to A, C, G and T, namely, 5-methylcytosine, in which a methyl group is attached to the fifth carbon of the cytosine ring (5-mC). 5-mC is always attached only to the C of a CG dinucleotide (5′-mCG-3′), which is frequently marked CpG. The C of CpG is mostly methylated by attachment with a methyl group. The methylation of this CpG inhibits a repetitive sequence in genomes, such as Alu or transposon, from being expressed. In addition, this CpG is a site where an epigenetic change in mammalian cells appears most often. The 5-mC of this CpG is naturally deaminated to T, and thus the CpG in mammal genomes shows only 1% of frequency, which is much lower than a normal frequency (¼×¼=6.25%).
Regions in which CpGs are exceptionally integrated are known as CpG islands. The term “CpG islands” refers to sites which are 0.2-3 kb in length, and have a C+G content of more than 50% and a CpG ratio of more than 3.75%. There are about 45,000 CpG islands in the human genome, and they are mostly found in promoter regions regulating the expression of genes. Actually, the CpG islands occur in the promoters of housekeeping genes accounting for about 50% of human genes (Cross, S. et al., Curr. Opin. Gene Develop., 5:309, 1995). It is known that aberrant DNA methylation occurs mainly in the 5′ regulatory region of the gene to reduce the expression of the gene (Kane M. F. et al., Cancer Res. 57(5):808-811). Herein, the 5′ regulatory region of the gene includes a promoter region, an enhancer region and a 5′ untranslated region. Recently, an attempt to examine the promoter methylation of tumor-related genes in blood, sputum, saliva, feces or urine and to use the examined results for the diagnosis and treatment of various cancers, has been actively conducted (Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60:892, 2000; Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000). Particularly, it is well known that DNAs are released from abnormal cells in the cancer tissue of cancer patients into blood by processes including apoptosis and necrosis, and thus exist as cell-free tumor DNA in the serum or plasma of the blood, and methylated DNA fragments are also present in the cell-free tumor DNA. The presence of this aberrant DNA methylation has been used as a marker for diagnosing cancer (deVos T. et al., Clin. Chem. 55:1337-1346; Potter N. T. et al., Clin. Chem. 60:9; Messaoudi S. EI. et al., Clinica Chimica Acta 424:222-230; Marzese D. M. et al., Expert Rev. Mol. Diagn. 13(8):827-844). Accordingly, the present inventors have conducted related studies, and have found that aberrant methylation of the syndecan-2 gene can be used specifically for diagnosis of colorectal cancer and this methylation is detected in serum (KR 10-1142131 B; Oh et al., J. Mol. Diag. 15(4):498-507).
However, KR 10-1142131 B does not disclose the use of DNA methylation to detect hyperplastic cells and polyps in the clone in a pre-cancerous stage by use of a cell-free DNA sample.
It is known that aberrant DNA methylation of a specific region of a gene among epigenetic changes may occur in a pre-cancerous stage, and in some cases, aberrant methylation of a specific DNA region may also occur in the pre-cancer stage, like hyperplastic cells, cell proliferative disorders and neoplasia.
Accordingly, the present inventors have made extensive efforts to develop a methylation marker capable of detecting a cell proliferative disorder corresponding to hyperplastic cells or polyps in the clone in a precancerous stage, and as a result, have found that the 5′ regulatory region of syndecan-2 gene (SDC2; NM_002998) is aberrantly methylated in colorectal tissue in a precancerous stage, and particularly, information for detection of persons having precancerous lesions can be provided by measuring frequent methylation in a cell-free DNA in the blood of these patients, thereby completing the present invention.
The information disclosed in the Background Art section is only for the enhancement of understanding of the background of the present invention, and therefore may not contain information that forms a prior art that would already be known to a person of ordinary skill in the art.